Pharmaceutical screens and biological assays have been used for decades in the pharmaceutical and biotech industries to identify lead compounds in the search for new pharmaceutical agents. In the last decade, the chemist""s ability to synthesize large numbers of chemical compounds in a short amount of time through techniques such as combinatorial chemistry has greatly increased (for a recent review of the area of combinatorial chemistry, please see Geysen et al. Molec. Immunol. 23:709-715, 1986; Houghton et al. Nature 354:84-86, 1991; Frank Tetrahedron 48:9217-9232, 1992; Bunin et al. Proc. Natl. Acad. Sci. USA 91:4708-4712, 1994; Thompson et al. Chem. Rev. 96:555-600, 1996; Keating et al. Chem. Rev. 97:449-472, 1997; Gennari et al. Liebigs Ann./Recueil 637-647, 1997; Reddington et al. Science 280:1735-1737, 1998; each of which is incorporated herein by reference), and it has expanded beyond the capacity of traditional screening methods. Often, thousands to millions of compounds need to be screened to identify those having a desired pharmaceutical property (e.g., anti-neoplastic activity, immunosuppressive activity, etc.). Many of the currently available screens are biochemical assay systems in which a compound is added to a purified or partially purified cell extract to see if it possesses the desired activity. In contrast to the biochemical assay systems, currently available cell-based assay systems identify bioactive molecules that are cell-permeable and work within physiological environments. However, one of the major drawbacks to cell-based assay screens is the high false-positive rate resulting from non-specific effects of the compound within the cell (for examples, please see Sarver et al. AIDS Res. Hum. Retroviruses 8:659-666, 1992; Witvrouw et al Antimicrob. Agents Chemother. 36:2628-2633, 1992; each of which is incorporated herein by reference). Given the fundamental importance of gene regulation in many disease states, one typical cell-based assay measures the activity of a reporter gene under the control of a specific reporter. However, inhibition of reporter gene expression does not necessarily indicate a specific interference with promoter activity but could reflect a non-specific inhibition of cellular functions, for example due to cytotoxicity.
One particularly important protein in the study of cancer is the nuclear phosphoprotein, p53. p53 is thought to be mutated in over 50% of human cancers. Mutations in the p53 gene have been found in tumors of colon, lung, breast, ovary, bladder, and several other organs. When mutant forms of the p53 gene are introduced into primary fibroblasts, these cells become immortalized. The wild type p53 gene has been shown to suppress the growth of transformed human cells, but oncogenic forms of p53 lose this suppressor function. Therefore, the p53 gene has been termed a xe2x80x9ctumor suppressorxe2x80x9d gene. Given the role of p53 in tumorigenesis, it has become an important potential target in the search for new anti-neoplastic agents.
The wild type p53 may be interfered with functionally. For example, a transforming viral infection of the cell can interfere with the p53 protein product. For instance, certain strains of human papillomavirus (HPV) are transforming and are known to interfere with the level of p53 protein in the infected cell because the virus produces a protein, E6, which promotes degradation of the p53 protein.
There is also an interest in p53 because p53 protein is capable of inducing apoptosis in certain cells. In apoptosis, or xe2x80x9cprogrammed cell deathxe2x80x9d, a series of lethal events for the cell appear to be generated directly as a result of transcription of cellular DNA. For example, lymphocytes exposed to glucocorticoids die by apoptosis. Involution of hormone sensitive tissue such as breast and prostate that occurs when the trophic hormone is removed occurs via apoptosis.
In particular, recent studies have indicated that the introduction of wild type (non-mutated) p53 into transformed cell lines that carry a mutant form of p53 induces the cells to undergo apoptosis with disintegration of nuclear DNA. It is believed that p53 may suppress tumor development by inducing apoptosis, thus modulating cell growth.
Given the importance of p53 in a variety of physiological and disease states, there is a need for cell-based assays with low backgrounds that could be used in screening compounds to identify inhibitors and activators of p53. Moreover, both the rapid increase of new drug targets through genomics research and the availability of vast libraries of chemical compounds create an enormous demand for new technologies which would improve the screening process.
The present invention provides assay systems for screening chemical compounds to identify activators and inhibitors of proteins of interest (e.g., transcription factors, enzymes, and tumor suppressors).
In one aspect, the invention provides a cell-based triple readout assay system for identifying compounds that affect transcriptional activity. Three separate cell lines, each containing a different engineered construct, are used. Two have been transfected with a construct comprising a reporter gene and a modulatable transcriptional regulatory sequence known to bind a selected transcription factor. Each of the two cell lines has a different reporter gene, and the construct is integrated into the genome in a different location to control for the effect of flanking sequences on the transcription of the reporter gene. A third cell line has been transfected with a construct comprising a third reporter gene operably linked to a constitutive promoter. The third cell line is used to assess general cytotoxicity of the test compound. At least one cell derived from each of the three cell lines is contacted with the test compound, and the levels of the reporter genes are assayed and used to determine the specificity of the test compound on the transcription factor or transcription factor pathway. In a particularly preferred embodiment, the transcription factor of interest is p53.
In another aspect, the invention provides a cell-based double readout assay system for identifying compounds that affect protein stability/levels in cells. A fusion protein is created between a protein of interest and a first reporter protein. The fusion protein and a second reporter protein are expressed in a cell line to which the test compound is added. The second reporter protein is used to control for non-specific effects such as cytotoxicity. The levels of the two proteins (i.e., the fusion protein and the second reporter protein) are measured to assess the specificity of the test compound on the protein of interest. In a particularly preferred embodiment, the two proteins are translated from the same mRNA transcript of an engineered DNA construct.
In a particularly preferred embodiment of this aspect of the present invention, the protein of interest is p53. One critical point of regulation of p53 occurs at the protein level. Tumor mutations that affect its conformation typically increase its half-life, in part by inhibiting its degradation by the ubiquitin-proteasome pathway. Consistent with its critical role in tumor suppression, many oncoproteins including human papillomavirus E6 oncoprotein target the p53 protein and alter its stability.
In yet another aspect, the invention provides chemical inhibitors and activators of p53. Such inhibitors and activators may preferably be identified and/or characterized using one or both of the inventive triple and double readout assay systems. In certain clinical situations, it is desirable to suppress the cellular effects of p53. For example, p53-dependent apoptosis is thought to contribute to the toxic side effects of anti-cancer treatment with chemotherapy. In certain preferred embodiments of the invention, the p53 inhibitors or activators are provided in the context of a pharmaceutical composition. In a preferred embodiment, the inhibitors and activators are small molecules.
In another aspect, the invention provides kits for performing the double and triple readout assays. Preferably, an inventive kit contains all the reagents needed to assay a test compound for its effect on transcription and/or protein stability/levels. In a particularly preferred embodiment, a kit to be used in performing the triple readout assay contains the three cell lines described above. A kit for performing the double readout assay preferably contains a cell line stably transfected with a fusion protein and a second reporter protein. In another preferred embodiment, an inventive kit comprises DNA constructs to be used in transfecting a cell line. Preferred inventive kits may also contain additional reagents such as media for growing the cells, enzyme substrates (e.g., the substrate of luciferase), DNA damaging compounds (e.g., adriamycin), human papillomavirus E6 oncoprotein, growth factors, etc.
Unless indicated otherwise, the terms defined below have the following meanings:
xe2x80x9cCompoundxe2x80x9d: The term xe2x80x9ccompoundxe2x80x9d or xe2x80x9cchemical compoundxe2x80x9d as used herein can include organometallic compounds, polynucleotides, oligonucleotides, peptides, proteins, organic compounds, metals, transitional metal complexes, and small molecules. In a particularly preferred embodiment, the term compound refers to small molecules (e.g., preferably, non-peptidic and non-oligomeric) and excludes peptides, polynucleotides, transition metal complexes, metals, and organometallic compounds.
xe2x80x9cConstitutive promoterxe2x80x9d: The term constitutive promoter refers to a promoter that is always xe2x80x9conxe2x80x9d. In other words, genes operably linked to a constitutive promoter are always being transcribed to produce mRNA.
xe2x80x9cConstructxe2x80x9d: The term construct refers to any polynucleotide that has been manipulated by the hand of man. Specifically, the construct is isolated from other sequences that are found in the natural state. The construct may be produced by recombinant known in the art such as the polymerase chain reaction. Preferably, the polynucleotide contains various elements that are operably linked, and the construct is introduced into a cell. For example, the construct may contain a promoter operably linked to a coding sequence, and the construct may be introduced into a cell to cause the cell to produce the encoded protein. In a preferred embodiment, the construct has been created or engineered by the hand of man and does not occur naturally.
xe2x80x9cFusion proteinxe2x80x9d: The term xe2x80x9cfusion proteinxe2x80x9d refers to a protein comprising two or more polypeptides that, although typically unjoined in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. The two or more polypeptide components can be either directly joined or indirectly joined through a peptide linker/spacer. The fusion protein may be translated by a ribosome from mRNA as a single polypeptide, or the polypeptides may be joined using synthetic or enzymatic chemistry.
xe2x80x9cModulatable transcriptional regulatory sequencexe2x80x9d: The term xe2x80x9cmodulatable transcriptional regulatory sequencexe2x80x9d refers to a DNA sequence capable of regulating the initiation of transcription from the promoter of the reporter gene by the binding of a protein to the sequence. The protein preferably binds a regulatory sequence of the construct in which the promoter, modulatable transcriptional regulatory sequence, and reporter gene are operably linked, and thereby the protein either up-regulates or down-regulates the transcription from the promoter.
xe2x80x9cOperably linkedxe2x80x9d: The term operably linked refers to two segments of polynucleotide sequence that can affect each other. In a particularly preferred embodiment, one of the two segments is a sequence that binds a protein (e.g., polymerase, enhancer, and transcription factor), and the binding of the protein to the sequence leads to the transcription of a gene sequence located in the second segment. In another particularly preferred embodiment, the binding of a molecule (e.g., nucleic acid, small molecule, protein, and peptide) to one segment may inhibit or enhance the binding of another molecule (e.g., nucleic acid, small molecule, protein, and peptide) to the second segment. Preferably, two operably linked segments are covalently linked, but any type of association sufficient to achieve the desired results is considered to be operably linked in the context of the present invention.
xe2x80x9cp53xe2x80x9d: The term xe2x80x9cp53xe2x80x9d as used in the present invention refers to both the gene and protein form of p53 or any homolog of p53 or member of the family of p53 genes. The homolog should be at least 50% homologous to the mouse p53 DNA or protein sequence; preferably, at least 60% homologous, and most preferably, greater than 75% homologous. A homolog of p53 may also be identified by its activity such as its ability to suppress the growth of transformed cells. In another preferred embodiment, the homolog of p53 is identified by its location in the genome (e.g., location on the chromosome). In yet another preferred embodiment, the homolog of p53 is able to hybridize to the p53 gene under standard hybridization conditions. p53 may also refer to a fragment of a p53 gene. In certain preferred embodiments, p63, p73, and homologs thereof are considered to be p53 family members. In other preferred embodiments, homologs are at least 50% homologous within the central sequence-specific DNA binding domain, the N-terminal transactivation domains, and/or the C-terminal oligomerization domain, more preferably greater than 60% homologous.
xe2x80x9cp53 binding elementxe2x80x9d: The term xe2x80x9cp53 binding elementxe2x80x9d refers to a sequence of a polynucleotide that binds the p53 protein. In a preferred embodiment, the p53 binding element is the p53 binding element found in the p21, bax, or 14-3-3 gene. The p53 binding element may comprise multiple binding elements (i.e., be multimerized).
xe2x80x9cPolynucleotidexe2x80x9d or xe2x80x9coligonucleotidexe2x80x9d: Polynucleotide or oligonucleotide refers to a polymer of nucleotides. Preferably, the polynucleotide comprises at least three nucleotides, more preferably it comprises at least 10 nucleotides, and most preferably it comprises at least 100 nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g, 2xe2x80x2-fluororibose, ribose, 2xe2x80x2-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5xe2x80x2-N-phosphoramidite linkages).
xe2x80x9cProteinxe2x80x9d: According to the present invention, a xe2x80x9cproteinxe2x80x9d comprises a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain;) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a hydroxyl group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
xe2x80x9cReporter genexe2x80x9d: As used herein, the term xe2x80x9creporter genexe2x80x9d refers to a gene whose transcript or any other gene product (e.g., protein) is detectable. Preferably, the gene product is also quantifiable. More preferably, the gene product is detectable using a standard assay. Most preferably, the gene product is detectable using a standard assay for which the reagents used in the assay are available in a kit. In certain preferred embodiments, the reporter gene encodes a fluorescent protein or an enzyme whose activity is detectable and preferably quantifiable.
xe2x80x9cSmall Moleculexe2x80x9d: As used herein, the term xe2x80x9csmall moleculexe2x80x9d refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are xe2x80x9cnatural product-likexe2x80x9d, however, the term xe2x80x9csmall moleculexe2x80x9d is not limited to xe2x80x9cnatural product-likexe2x80x9d compounds. Rather, a small molecule is typically characterized in that it contains several carbonxe2x80x94carbon bonds, and has a molecular weight of less than 1500, although this characterization is not intended to be limiting for the purposes of the present invention. Examples of xe2x80x9csmall moleculesxe2x80x9d that occur in nature include, but are not limited to, taxol, dynemicin, and rapamycin. Examples of xe2x80x9csmall moleculesxe2x80x9d that are synthesized in the laboratory include, but are not limited to, compounds described in Tan et al., (xe2x80x9cStereoselective Synthesis of over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assaysxe2x80x9d J. Am. Chem. Soc. 120:8565, 1998) and pending application Ser. No. 08/951,930 xe2x80x9cSynthesis of Combinatorial Libraries of Compounds Reminiscent of Natural Productsxe2x80x9d, the entire contents of which are incorporated herein by reference.